AFM is a useful tool, not only to be able to see the walker, but also to

+

-

image origami and make sure it is well-formed when certain parameters are

+

[[Image:PT_Ori.PNG|thumb|center|800px|Figure 1. The Random Walking Playground used for AFM experiments. The dark blue dots represent track 1 strands. The light blue dots represent track 2 strands. The light blue dots in the upper left corner are hairpin markers used to tell the orientation of origami in AFM. The green dot is a biotinylated control staple that was used in experiments after the walker could not be observed to see what streptavidin looks like when it is on origami.]]<br>

-

changed. It is arguably the best way to debug any origami-related problems

+

-

the system may have, since you can see the origami, and potentially see

+

-

problems it may have. Thus, we began by imaging origami ([[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Probes|Figure 2]]) with

+

AFM is a useful tool, not only to be able to see the walker, but also to image origami and make sure it is well-formed when certain parameters are changed. It is arguably the best way to debug any origami-related problems the system may have, since you can see the origami, and potentially see problems it may have. Thus, we began by imaging origami ([[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Probes|Figure 4]]) with

probes laid out as shown in the random walking playground. In a few of the rectangles, the probes

probes laid out as shown in the random walking playground. In a few of the rectangles, the probes

can be seen as a faint line across the diagonal, but a more obvious feature is

can be seen as a faint line across the diagonal, but a more obvious feature is

-

many distorted and/or destroyed origami. [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Tracks|Figure 3]] shows origami that has

+

many distorted and/or destroyed origami. [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Tracks|Figure 5]] shows origami that has

tracks in addition to probes, where the tracks can be seen as a clear diagonal

tracks in addition to probes, where the tracks can be seen as a clear diagonal

line on the origami, with much fewer distorted/destroyed origami.

line on the origami, with much fewer distorted/destroyed origami.

-

To image the walker, we ordered walkers with biotin attached at the 3’

+

To image the walker, we ordered walkers with biotin attached at the 5’

-

end (the top of the walker). A streptavidin molecule can bind to up to

+

end (the top of the walker) as drawn in Figure 2a. A streptavidin molecule can bind to up to

four biotin molecules, and since streptavidin is fairly large, it can be seen

four biotin molecules, and since streptavidin is fairly large, it can be seen

as a bright spot under AFM, which will contrast it to the darker origami.

as a bright spot under AFM, which will contrast it to the darker origami.

Unfortunately, when we added the biotinylated walker start complex to the

Unfortunately, when we added the biotinylated walker start complex to the

-

origami, nothing could be seen ([[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Unobservable Walkers|Figure 4]]). Various hypotheses were formed and tested

+

origami, nothing could be seen ([[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Unobservable Walkers|Figure 6]]). Various hypotheses were formed and tested

One hypothesis is that the streptavidin stock was bad or for some reason was not binding properly to DNA.

One hypothesis is that the streptavidin stock was bad or for some reason was not binding properly to DNA.

-

To prove this was not the case, we ordered a biotinylated staple (orange

+

To prove this was not the case, we ordered a biotinylated staple (green

-

dot in the random walking playground). The streptavidin was seen as a very bright spot on most

+

dot in Figure 1). The streptavidin was seen as a very bright spot on most

origami. We decided to use the staple in all future AFM experiments as

origami. We decided to use the staple in all future AFM experiments as

-

a control. A second hypothesis was that the insertion mechanism was not

+

a control.

+

+

A second hypothesis was that the insertion mechanism was not

working, and that the walker was ﬂoating in solution. This seems unlikely,

working, and that the walker was ﬂoating in solution. This seems unlikely,

because free ﬂoating wakers could diﬀuse to the goal when triggered, but

because free ﬂoating wakers could diﬀuse to the goal when triggered, but

Line 31:

Line 35:

another biotinylated staple at SP10. The streptavidin can be seen but less

another biotinylated staple at SP10. The streptavidin can be seen but less

frequently than the biotinylated control staple. This low frequency could

frequently than the biotinylated control staple. This low frequency could

-

simply be due to stoichiometric diﬀerences, since in [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Streptavidin Control|Figure 5]]

+

simply be due to stoichiometric diﬀerences, since in [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Streptavidin Control|Figure 7]]

we see that the control staple appears on almost all origami, whereas before it was absent

we see that the control staple appears on almost all origami, whereas before it was absent

-

in many. A third hypothesis was that the streptavidin is too high from the

+

in many.

+

+

A third hypothesis was that the streptavidin is too high from the

surface on DNA with multiple nicks that could easily “dodge” the AFM tip.

surface on DNA with multiple nicks that could easily “dodge” the AFM tip.

A proposed solution for this was to order walkers biotinylated at the 3’ end

A proposed solution for this was to order walkers biotinylated at the 3’ end

-

instead, so it would only be 20 base pairs away from the surface. When this

+

instead (Figure 2a), so it would only be 20 base pairs away from the surface. When this

was attempted, the walker was still not seen. One possibility was that the

was attempted, the walker was still not seen. One possibility was that the

streptavidin was there but since it was still far from the surface, its brightness

streptavidin was there but since it was still far from the surface, its brightness

Line 43:

Line 49:

the walker (biotinylated at the 3’ end) onto the origami, rather than inserting

the walker (biotinylated at the 3’ end) onto the origami, rather than inserting

it. Surprisingly, several bright spots were seen where the walker should be

it. Surprisingly, several bright spots were seen where the walker should be

-

([[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Observable Annealed Walkers|Figure 6]]). The origami that may have visible streptavidin are circled in

+

([[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Observable Annealed Walkers|Figure 8]]). The origami that may have visible streptavidin are circled in

-

blue. [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Observable Annealed Walkers|Figure 6b]] shows the same area imaged after [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Observable Annealed Walkers|Figure 6a]], and we see

+

blue. [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Observable Annealed Walkers|Figure 8b]] shows the same area imaged after [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Observable Annealed Walkers|Figure 8a]], and we see

that two of the origami with potential visible walkers still had the same spot.

that two of the origami with potential visible walkers still had the same spot.

This could suggest that the problem is not that the walker “dodges” the tip,

This could suggest that the problem is not that the walker “dodges” the tip,

Line 61:

Line 67:

more potential visible walkers were seen, as well as more unexplainable spots.

more potential visible walkers were seen, as well as more unexplainable spots.

The most likely hypothesis is still that the streptavidin moves too much to

The most likely hypothesis is still that the streptavidin moves too much to

-

be caught by the tip. To counter this, we are currently trying to lock down

+

be caught by the tip.

+

+

To counter this, we are currently trying to lock down

the walker at a given position using the surrounding tracks, and the fact that

the walker at a given position using the surrounding tracks, and the fact that

-

multiple biotin molecules can be bound to one streptavidin.

+

multiple biotin molecules can be bound to one streptavidin. A [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Walker Lock-Down Protocol | protocol]] was developed

+

to lock down the walker, using inspiration from the spider walker [1] and suggestions from Jongmin Kim to try making such a walker. Using this protocol, we were able to see the walker ([[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/AFM_Experiments#Origami with Observable Walkers on Tracks | Figure 9]])!

+

+

==Regular Rectangular Origami==

==Regular Rectangular Origami==

-

[[Image:Regular_rectangle.bmp|Figure 1. We started by trying to form a regular rectangle to make sure our protocols worked as expected.]]<br>We started by trying to form a regular rectangle to make sure our protocols worked as expected.<br><br>

+

+

We started by trying to form a regular rectangle to make sure our protocols worked as expected.

[[Image:Modified_rectangle.bmp|Figure 2. We reformed the regular rectangle using our modified staple strands to show that the probes don't interfere with the binding in the rectangle.]]<br>We reformed the regular rectangle using our modified staple strands to show that the probes don't interfere with the binding in the rectangle.<br><br>

+

We reformed the regular rectangle using our modified staple strands (at the locations we would put the tracks in Figure 1) to show that the probes don't interfere with the binding in the rectangle.

[[Image:No_walker_1.bmp|Figure 4. We attempted to insert our walker at the beginning of the track for the first time, but we can't see it on the AFM images. This could be due to any one of a number of issues.]]<br>We attempted to insert our walker at the beginning of the track for the first time, but we can't see it on the AFM images. This could be due to any one of a number of issues.<br><br>

+

+

We attempted to insert our walker at the beginning of the track for the first time, but we can't see it on the AFM images. This could be due to any one of a number of issues as discussed above.

[[Image:Strep_control.bmp|Figure 5. We first see what our walker should look like by adding another control to our origami. The new bright dot is the protein streptavidin.]]<br>We first see what our walker should look like by adding another control to our origami. The new bright dot is the protein streptavidin.<br><br>

+

We first see what our walker should look like by adding another control to our origami. The new bright dot is the biotinylated control staple (green dot in Figure 1) bound to streptavidin, which is tested by a biotinylated staple as discussed above.

[[Image:Annealedwalkers.png|Figure 6. After some work, we can now see walkers on the surface of our origami.]]<br>After some work, we can now see walkers on the surface of our origami.<br><br>

+

Successful images showing that walkers are landing on the surface. Consecutive images of origami with no probes, except for the one the walker is attached to. Blue circles indicate origami with potential visible walkers. Red circle shows origami with a potential walker in the wrong position.

Successful images showing that walkers are landing on the surface with tracks. Blue circles indicate tracks that we speculate have the walker at the starting position. Red circles indicate tracks that we speculate have the walker, but either at the wrong position or at multiple positions, perhaps due to some remaining free-floating streptavidin.

Here we provide a high level overview of the protocol we use to lock-down the walker in place. For a more detailed description of the protocol see [[Biomod/2011/Caltech/DeoxyriboNucleicAwesome/Protocols#Walker Lock-Down Protocol|protocol]] page.

+

Essentially, we want to take advantage of the fact that up to four biotins can bind to a single streptavidin protein. Since there are tracks surrounding the walker (regardless of the walker's position), if we place biotinylated walkers on those surrounding tracks, they can bind to the same streptavidin as the original walker. Thus, we can form spiders [1] that are less likely to dodge the AFM tip since they are not as floppy. We can clearly add walkers such that they bind to every track in solution to achieve this, but since there is excess streptavidin in solution, all walkers that land on origami can form spiders with their neighbors, which means we should expect the entire track to be bright, rather than just the position of the original walker. Thus, before adding the extra biotinylated walkers, we must first wash the buffer on the mica to remove all excess streptavidin, so the only spiders that form are those involving the original walkers. This protocol proved to be successful using 3'-end biotinylated walkers, but so far has not worked using 5'-end biotinylated walkers.

Figure 1. The Random Walking Playground used for AFM experiments. The dark blue dots represent track 1 strands. The light blue dots represent track 2 strands. The light blue dots in the upper left corner are hairpin markers used to tell the orientation of origami in AFM. The green dot is a biotinylated control staple that was used in experiments after the walker could not be observed to see what streptavidin looks like when it is on origami.

AFM is a useful tool, not only to be able to see the walker, but also to image origami and make sure it is well-formed when certain parameters are changed. It is arguably the best way to debug any origami-related problems the system may have, since you can see the origami, and potentially see problems it may have. Thus, we began by imaging origami (Figure 4) with
probes laid out as shown in the random walking playground. In a few of the rectangles, the probes
can be seen as a faint line across the diagonal, but a more obvious feature is
many distorted and/or destroyed origami. Figure 5 shows origami that has
tracks in addition to probes, where the tracks can be seen as a clear diagonal
line on the origami, with much fewer distorted/destroyed origami.
To image the walker, we ordered walkers with biotin attached at the 5’
end (the top of the walker) as drawn in Figure 2a. A streptavidin molecule can bind to up to
four biotin molecules, and since streptavidin is fairly large, it can be seen
as a bright spot under AFM, which will contrast it to the darker origami.
Unfortunately, when we added the biotinylated walker start complex to the
origami, nothing could be seen (Figure 6). Various hypotheses were formed and tested
as to why the walker could not be seen.

One hypothesis is that the streptavidin stock was bad or for some reason was not binding properly to DNA.
To prove this was not the case, we ordered a biotinylated staple (green
dot in Figure 1). The streptavidin was seen as a very bright spot on most
origami. We decided to use the staple in all future AFM experiments as
a control.

A second hypothesis was that the insertion mechanism was not
working, and that the walker was ﬂoating in solution. This seems unlikely,
because free ﬂoating wakers could diﬀuse to the goal when triggered, but
the ﬂuorescence experiments showed that in the absence of tracks, walkers
did not reach the goal. In any case, this hypothesis was tested by inserting
another biotinylated staple at SP10. The streptavidin can be seen but less
frequently than the biotinylated control staple. This low frequency could
simply be due to stoichiometric diﬀerences, since in Figure 7
we see that the control staple appears on almost all origami, whereas before it was absent
in many.

A third hypothesis was that the streptavidin is too high from the
surface on DNA with multiple nicks that could easily “dodge” the AFM tip.
A proposed solution for this was to order walkers biotinylated at the 3’ end
instead (Figure 2a), so it would only be 20 base pairs away from the surface. When this
was attempted, the walker was still not seen. One possibility was that the
streptavidin was there but since it was still far from the surface, its brightness
would blend in with that of tracks. Thus, origami was formed without any
probes except for SP10. Since this was the only probe, we could now anneal
the walker (biotinylated at the 3’ end) onto the origami, rather than inserting
it. Surprisingly, several bright spots were seen where the walker should be
(Figure 8). The origami that may have visible streptavidin are circled in
blue. Figure 8b shows the same area imaged after Figure 8a, and we see
that two of the origami with potential visible walkers still had the same spot.
This could suggest that the problem is not that the walker “dodges” the tip,
but rather that for some other reason most origami do not have a walker
start complex with streptavidin. However, since one of the circled origami
(the top right one) does not seem to have the bright spot again, that is not
necessarily the case. It could be that on the origami with visible walkers,
the walkers somehow got stuck in some position, so the streptavidin can no
longer move. The red circled origami also contains a bright spot on the side
opposite the control staple and marker that appears in both images, but this
spot is neither in the right position nor anywhere on the track. This could
also be an instance of a walker that got stuck in some position on the origami
(perhaps after being dismantled from its original position). To make matters
more confusing, to the right of the bottommost blue circle are two origami
that appear to have three bright spots all on the same side. In other images,
more potential visible walkers were seen, as well as more unexplainable spots.
The most likely hypothesis is still that the streptavidin moves too much to
be caught by the tip.

To counter this, we are currently trying to lock down
the walker at a given position using the surrounding tracks, and the fact that
multiple biotin molecules can be bound to one streptavidin. A protocol was developed
to lock down the walker, using inspiration from the spider walker [1] and suggestions from Jongmin Kim to try making such a walker. Using this protocol, we were able to see the walker ( Figure 9)!

Regular Rectangular Origami

We started by trying to form a regular rectangle to make sure our protocols worked as expected.

Origami with Probes

We reformed the regular rectangle using our modified staple strands (at the locations we would put the tracks in Figure 1) to show that the probes don't interfere with the binding in the rectangle.

Figure 4. AFM image of origami with probes

Origami with Tracks

Diagonal tracks were well formed on the origami as shown in the figure.

Figure 5(1). AFM image of origami with tracks

Figure 5(2). AFM image of origami with tracks. Magnified

Origami with Unobservable Walker

We attempted to insert our walker at the beginning of the track for the first time, but we can't see it on the AFM images. This could be due to any one of a number of issues as discussed above.

Figure 6. Origami with Unobservable Walker

Origami with Streptavidin Control

We first see what our walker should look like by adding another control to our origami. The new bright dot is the biotinylated control staple (green dot in Figure 1) bound to streptavidin, which is tested by a biotinylated staple as discussed above.

Figure 7. Origami with Streptavidin Control

Origami with Observable Annealed Walkers

Successful images showing that walkers are landing on the surface. Consecutive images of origami with no probes, except for the one the walker is attached to. Blue circles indicate origami with potential visible walkers. Red circle shows origami with a potential walker in the wrong position.

Figure 8. Origami with Observable Annealed Walkers

Origami with Observable Walkers on Tracks

Successful images showing that walkers are landing on the surface with tracks. Blue circles indicate tracks that we speculate have the walker at the starting position. Red circles indicate tracks that we speculate have the walker, but either at the wrong position or at multiple positions, perhaps due to some remaining free-floating streptavidin.

Figure 9.Origami with Observable Walkers on Tracks

Walker Lock-Down Protocol

Here we provide a high level overview of the protocol we use to lock-down the walker in place. For a more detailed description of the protocol see protocol page.
Essentially, we want to take advantage of the fact that up to four biotins can bind to a single streptavidin protein. Since there are tracks surrounding the walker (regardless of the walker's position), if we place biotinylated walkers on those surrounding tracks, they can bind to the same streptavidin as the original walker. Thus, we can form spiders [1] that are less likely to dodge the AFM tip since they are not as floppy. We can clearly add walkers such that they bind to every track in solution to achieve this, but since there is excess streptavidin in solution, all walkers that land on origami can form spiders with their neighbors, which means we should expect the entire track to be bright, rather than just the position of the original walker. Thus, before adding the extra biotinylated walkers, we must first wash the buffer on the mica to remove all excess streptavidin, so the only spiders that form are those involving the original walkers. This protocol proved to be successful using 3'-end biotinylated walkers, but so far has not worked using 5'-end biotinylated walkers.